Increased NMDAR1 Subunit mRNA Levels In Purkinje Cells In the Crus II Cerebellar Hemisphere Region In Autism: An In Situ Hybridization Study

Background: Neurochemical studies on affected cell types and their associated receptors have consistently shown significant changes in the GABAergic system in the autism postmortem brain. In contrast, there is a paucity of information on the excitatory glutamatergic system in autism. An earlier study in the postmortem cerebellum reported a significant increase in the protein levels of the NMDAR1 gene in autism compared to controls and normal density of NMDA receptors in the cerebellar molecular and granular layers in the autism group (Purcell et al., 2001, Neurology 57:1618-1628). It is hypothesized that there may be an imbalance between the inhibitory GABAergic and excitatory glutamatergic systems in the autism brain, which provides a potential pathway(s) for the affected motor and/or cognitive deficits. The posterior lateral cerebellar hemisphere (Crus II) is an ideal substrate to investigate changes in glutamatergic receptor mRNA levels in autism due to a previously reported decrease in GAD 67 mRNA levels in Purkinje cells (PCs), as well as marked deficits in Purkinje cell number.

Objectives: To elucidate the state of NMDAR1 receptor subunit mRNA levels in Purkinje cells in the Crus II region in postmortem adult autism cases, compared to age- and post-mortem interval matched controls.

Methods: In situ hybridization histochemistry and autoradiography were used to examine the cellular distribution of NMDAR1 receptor subunit mRNA in the Crus II region and quantitative analysis of NMDAR1 expression was obtained by measuring silver grain density in 120 Purkinje cell somata for each case. Grain density corresponding to NMDAR1 mRNA labeling was expressed as a mean number of pixels per surface area using NIH Image J analysis software and two-tailed unpaired t-tests were applied to determine significance at a P<0.05 level.

Results: Based on the initial analysis of 6 autism and 6 control cases, significant increases in NMDAR1 mRNA levels were found in PCs in the autism group compared to controls. Quantification of the NMDAR1 labeling revealed a 21.9% signal increase in PCs in the autism group when compared with controls (11.6±1.05 pixels/surface area in the autism group compared to 9.06± 0.45 in the control group; P=0.046 two-tailed unpaired t-test). Note that additional cases are currently being quantified and will be added to this data set to total 12 cases per group.

Conclusions: Results from the data thus far support the hypothesis that there are changes in the glutamate system in the cerebellum in autism and may contribute, along with previously reported alterations in GABA biomarkers, to the core neural features of the disorder.

Acknowledgements: Human tissue was obtained from the Autism Tissue Program and The Autism Research Foundation via the Harvard Brain Tissue Resource Center, and from the NICHD Brain and Tissue Bank for Developmental Disorders at the University of Maryland, Baltimore.